EP3334537B1 - Method for coating samples - Google Patents
Method for coating samples Download PDFInfo
- Publication number
- EP3334537B1 EP3334537B1 EP16717136.2A EP16717136A EP3334537B1 EP 3334537 B1 EP3334537 B1 EP 3334537B1 EP 16717136 A EP16717136 A EP 16717136A EP 3334537 B1 EP3334537 B1 EP 3334537B1
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- Prior art keywords
- gas
- spraying device
- housing
- coat
- solution
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- 238000000034 method Methods 0.000 title claims description 37
- 239000011248 coating agent Substances 0.000 title claims description 7
- 238000000576 coating method Methods 0.000 title claims description 7
- 239000007788 liquid Substances 0.000 claims description 34
- 239000007921 spray Substances 0.000 claims description 23
- 238000005507 spraying Methods 0.000 claims description 17
- 238000006073 displacement reaction Methods 0.000 claims description 2
- 239000007789 gas Substances 0.000 description 29
- 239000011159 matrix material Substances 0.000 description 22
- 239000013078 crystal Substances 0.000 description 16
- 239000010453 quartz Substances 0.000 description 12
- 239000000523 sample Substances 0.000 description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 12
- 238000000605 extraction Methods 0.000 description 8
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 4
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- 239000004696 Poly ether ether ketone Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 229920002530 polyetherether ketone Polymers 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000000859 sublimation Methods 0.000 description 3
- 230000008022 sublimation Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
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- 102000004190 Enzymes Human genes 0.000 description 1
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- 241000589614 Pseudomonas stutzeri Species 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
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- 210000004027 cell Anatomy 0.000 description 1
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- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 210000005260 human cell Anatomy 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000001871 ion mobility spectroscopy Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- -1 lipids Chemical class 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
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- 229920001296 polysiloxane Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
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Images
Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05B—SPRAYING APPARATUS; ATOMISING APPARATUS; NOZZLES
- B05B7/00—Spraying apparatus for discharge of liquids or other fluent materials from two or more sources, e.g. of liquid and air, of powder and gas
- B05B7/02—Spray pistols; Apparatus for discharge
- B05B7/06—Spray pistols; Apparatus for discharge with at least one outlet orifice surrounding another approximately in the same plane
- B05B7/062—Spray pistols; Apparatus for discharge with at least one outlet orifice surrounding another approximately in the same plane with only one liquid outlet and at least one gas outlet
- B05B7/066—Spray pistols; Apparatus for discharge with at least one outlet orifice surrounding another approximately in the same plane with only one liquid outlet and at least one gas outlet with an inner liquid outlet surrounded by at least one annular gas outlet
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
- H01J49/0409—Sample holders or containers
- H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05B—SPRAYING APPARATUS; ATOMISING APPARATUS; NOZZLES
- B05B5/00—Electrostatic spraying apparatus; Spraying apparatus with means for charging the spray electrically; Apparatus for spraying liquids or other fluent materials by other electric means
- B05B5/025—Discharge apparatus, e.g. electrostatic spray guns
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05B—SPRAYING APPARATUS; ATOMISING APPARATUS; NOZZLES
- B05B7/00—Spraying apparatus for discharge of liquids or other fluent materials from two or more sources, e.g. of liquid and air, of powder and gas
- B05B7/16—Spraying apparatus for discharge of liquids or other fluent materials from two or more sources, e.g. of liquid and air, of powder and gas incorporating means for heating or cooling the material to be sprayed
- B05B7/1606—Spraying apparatus for discharge of liquids or other fluent materials from two or more sources, e.g. of liquid and air, of powder and gas incorporating means for heating or cooling the material to be sprayed the spraying of the material involving the use of an atomising fluid, e.g. air
- B05B7/1613—Spraying apparatus for discharge of liquids or other fluent materials from two or more sources, e.g. of liquid and air, of powder and gas incorporating means for heating or cooling the material to be sprayed the spraying of the material involving the use of an atomising fluid, e.g. air comprising means for heating the atomising fluid before mixing with the material to be sprayed
Definitions
- the present invention relates to a method for coating samples by spraying them with a solution using a spray device, in which the sample (112) is sprayed several times in succession, a subsequent spray application only being carried out when the previously applied layer has dried .
- MALDI Imaging M atrix A ssisted L aser D esorption onisation I.
- MALDI Imaging She has taken on a pioneering role in the field of imaging mass spectrometry, which has been established in cancer research, pharmaceutical development and protein research for around 10 years.
- the advantage of the MALDI technique is that the tissue samples can be prepared for the examination without great effort, whereby not only freshly frozen samples, but also samples fixed by formalin and embedded in silicone and thus practically indefinitely durable for this new and very sensitive examination method are accessible, as are, for example, very old sample material, which can be over 100 years old.
- the basis of the MALDI technique is that the tissue sections are placed on steel plates known as targets or on microscope slides, on which they adhere permanently and fix themselves after drying. Subsequently, the cuts are coated with a mostly polar organic compound called a matrix, which has the property that it changes when it is irradiated with laser beams absorb the light in the UV range and then evaporate due to the high energy input. In the process, substances in the tissue sample are also dragged into the vapor phase that are actually not vaporizable at all and would burn if heated normally. Due to the extremely rapid evaporation, however, it is possible to evaporate such samples. These molecules are ionized in an electric field and recorded by a mass spectrometer. In these mass spectrometers they are then separated according to charge and size, with software decoding and identifying the composition from these two parameters.
- Another disadvantage is that a complete area is coated with the matrix next to the sample and not just the sample itself, which results in a high consumption of matrix solution, which is particularly important in the case of expensive matrices. In any case, only a few matrices are suitable for this method, while it cannot be used, for example, for enzyme solutions.
- the modified airbrush method should be emphasized, in which an optimized spray head is charged with an exact amount of liquid per unit of time. Otherwise, such a spray head, as the name suggests, uses airbrush technology, in which the air flow according to the ejector principle entrains the liquid and distributes it finely. Such a method is very suitable for extraction, the crystal sizes that can be achieved after the matrix has dried off in the range of 30 to 100 ⁇ m.
- the resolution in MALDI imaging is determined by the crystal size of the matrix and the laser diameter. The smaller the resolution of these two parameters, the more detailed information can be obtained about the tissue, e.g. B. liver, kidney, skin and brain as well as all other internal organs or their fine structure. This enables a wealth of new information, for example about the development of a cancer or the new compounds that are created by the cancer, which in turn enables the disease to be detected early.
- the object of the present invention is to improve a method of the type mentioned at the outset such that smaller Maxtrix crystals can be formed in the coating after it has dried.
- the coating should also be designed as homogeneously as possible in order to to enable the detection of the molecules not only qualitatively but also semi-quantitative or quantitative.
- the object is achieved by a method of the type mentioned at the outset, in which the first applied layer is sprayed with a smaller amount of solution per area, the amount in the subsequent layers is increased until a certain amount of solution per area is reached is applied repeatedly for the last layer or for several layers.
- a preferred embodiment of the method provides that the first applied layer is sprayed with a smaller amount of solution per area, the amount is increased in the subsequent layers until a certain amount of matrix per area is reached, and this amount of solution is applied several times per area for the last layers.
- a typical number of layers is in the range between 5 to 10, for example 8 Layers, so that overall an appropriate thickness of the matrix results, which when irradiated with the laser then evaporates locally on an area reduced to the crystal size achieved.
- the first 1-3 layers, which are sprayed with the smaller amount of solution per area dry very quickly, so that no changes in the position of small molecules are to be feared.
- the amount can be doubled when applying the second layer and tripled when applying the third layer until a maximum is reached in the third to sixth layers that is applicable to all subsequent layers Application comes.
- the spray device formed in the usual way with an appropriate geometry of the spray head has an area with a diameter of e.g. 2 mm can be sprayed, it is expedient for correspondingly larger samples that this is sprayed line by line using a device with adjustability in the X and Y directions for applying a layer. After the sample has been sprayed line by line and a layer has been completely applied, the spray head then moves back to the starting position and repeats the application line by line for the next layer.
- a preferred further development of the invention provides for the use of a spray device in which the liquid outlet is provided at the end of a capillary line which, for example, protrudes centrally beyond the gas outlet.
- air or nitrogen are suitable as gases, depending on the type of matrix solution to be sprayed and the sample to be examined.
- the end of the capillary line is tapered on its outer circumference, preferably is tapered conically. It has been shown that this measure enables particularly fine droplet formation because the tapering, preferably in a conical shape, deflects the swirled gas flow particularly effectively in the direction of the liquid outlet.
- the gas outlet is formed by an annular gap between the outer wall of the capillary line and a guide hose or tube.
- the guide tube or the guide tube also serves to stabilize the sensitive capillary line, which is usually designed as a quartz capillary.
- the guide tube or the guide tube which can for example consist of PEEK (polyether ether ketone), can in turn be stabilized by a larger diameter, with the guide tube and / or the capillary line preferably being held gas-tight in a housing.
- the housing has the task of precisely defining the position of the nozzle head and, on the other hand, the housing preferably has a gas connection for the gas supply, with a gas passage in the housing in an annular gap between the guide tube or the guide tube and the capillary line. In this way, the function of the housing is to guide the gas flow from the gas supply into the annular gap between the guide tube or the guide tube and the capillary line.
- the capillary line protrudes through the housing and is connected at its other end to an already mentioned feed pump for the supply of liquid, whereby a dispenser with a suitable high resolution, for example 24,000 steps per syringe filling, can be used instead of a pump.
- a plug is preferably provided on the side of the housing facing away from the spray head, which plug closes the housing in a gastight manner against the outer wall of the capillary line.
- the housing preferably has a shoulder which is arranged at a defined distance from the liquid outlet. The exact position of the liquid outlet defines the position of the nozzle head.
- the nozzle head is preferably held on a displacement device which enables the position of the nozzle head to be moved in the X, Y and / or Z directions. Basically, the ability to move in the X and Y directions is more important, since it may also have a fixed position, i.e. H. a fixed distance between the liquid outlet and the sample surface can be used.
- the nozzle head is brought into a defined position with respect to the tissue surface, for example with the aid of the paragraph described above on the housing.
- a nozzle head 10 is shown, which can also be referred to as a spray head.
- the nozzle head 10 is connected via a rear end 12 of a quartz capillary 14 as a capillary line to a liquid supply, which is not shown in detail.
- This hydration can be through a constantly pumping syringe pump or by a dispenser with a very high resolution of 24,000 steps per syringe filling in order to allow the most exact amounts of liquid possible through the capillary line of the quartz capillary 14 to a liquid outlet 16 (see also Fig. 2 ) to promote.
- the quartz capillary 14 is passed through a housing 18 which is closed at its rear end with the aid of a union nut 20, the union nut 20 engaging in a thread 22 in the housing 18 and sealing the housing 18 against the outer circumference of the quartz capillary 14.
- the housing itself is stepped with a shoulder 24, the function of which will be discussed in more detail later.
- the housing is provided on the inside with a central bore 26 which is in communication with a radial gas connection 28 which is to be connected to a gas supply (not shown).
- the gas supply provides for the supply of air or another suitable gas such as e.g. B. nitrogen, under a pressure of usually 2 to 3 bar, which is kept constant during operation, other pressure values can also be achieved.
- a first guide tube 32 is seated in an enlarged bore section 30, which guide tube can also be designed as a guide tube, within which a further guide tube 34 is let in in a pressure-tight manner.
- the second guide tube 34 encloses the quartz capillary 14 on its outer circumference with an annular gap, ie there is a gas passage between the second guide tube 34 and the quartz capillary between the bore 26 of the housing 10 and a gas outlet 36 (see Fig. 2 ) at the end of the second guide tube 34, in the middle of which the quartz capillary 14 protrudes a certain distance beyond the end of the second guide tube 34.
- the annular gap 38 is provided to blow out the gas supplied through the gas connection 28 at the end of the second guide hose 34, which is indicated by the arrows.
- the liquid outlet 16 takes place at a distance from the gas outlet 36 and is illustrated by the drops outlined. Due to the distance between the gas outlet 36 and the liquid outlet 16, the gas flow can swirl slightly, the end of the glass capillary 14 being provided with a conical taper 40 which promotes the swirled gas flow in the direction of the metered flow of liquid emerging from the liquid outlet 16.
- This conical tapering which can also be spherical, results in particularly fine droplet formation, the gas flow conveying the fine liquid droplets further in the direction of a sample arranged below the liquid outlet 16.
- the in Fig. 1 and 2 The nozzle head shown is mounted in a spray device 100, the shoulder 24 resting in a defined position of a receptacle so that the liquid outlet 16 is at a defined distance from metallic targets 110 arranged on a table on which tissue to be sprayed in preparation for further examinations 112 is applied.
- the receptacle 102 of the nozzle head 10 is arranged laterally movable in a Y-direction on a carrier 104, which in turn is attached to a rail 106 such that it can be moved in an X-direction, so that the nozzle head is adjustable by moving the receptacle 102 on the carrier 104 and moving the carrier 104 on the rail 106 in the X and Y directions.
- the receptacle 102 can be adjustable in the Z-direction in order to be able to set the distance between the liquid outlet 16 and the targets 110 or prepared glass carriers. As a rule, however, it is not necessary to adjust the distance between the spray head 10 and the targets 110 during the actual spraying process.
- the fabric 112 is normally sprayed in such a way that the nozzle head 10 scans the area to be sprayed line by line, since the area covered by the spray jet is usually smaller than the area of the fabric to be sprayed.
- the sprayed matrix solution covers a strip-shaped area with a width of approximately 2 mm.
- a matrix solution was sprayed which was mixed in the area of the nozzle head 10 with an air stream fed in at a constant pressure of 2.5 bar.
- the outer diameter of the selected quartz capillary was 280 ⁇ m, with an annular gap 60 ⁇ m high between the second guide tube 34 and the outer diameter of the quartz capillary 14, ie the inner diameter of the second guide tube made of PEEK was 400 ⁇ m.
- the quartz capillary 14 had an inner channel 42 with a diameter of 75 ⁇ m.
- a second layer was applied by spraying, which was carried out with otherwise the same process parameters while doubling the liquid supply to 20 ⁇ l per minute.
- a third layer was applied at 30 ⁇ l per minute, a fourth at 40 ⁇ l per minute, a total of 8 layers being applied, all of which were applied from the fourth layer onwards with a liquid supply of 40 ⁇ l per minute.
- the exemplary embodiment described, with its key data, is not mandatory.
- the dimensions of the quartz capillary and the guide tube can differ significantly, but the amount of liquid can also differ significantly from the stated values depending on the type of matrix solution to be sprayed. Deviations from the selected pressure are also possible with the gas supply in order to be able to apply different layers depending on the desired examination.
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- Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Sampling And Sample Adjustment (AREA)
Description
Die vorliegende Erfindung befasst sich mit einem Verfahren zum Beschichten von Proben durch Besprühen mit einer Lösung unter Verwendung einer Sprühvorrichtung, bei welchem die Probe (112) nacheinander mehrfach besprüht wird, wobei ein nachfolgender Sprühauftrag erst dann durchgeführt wird, wenn die zuvor aufgetragene Schicht abgetrocknet ist.The present invention relates to a method for coating samples by spraying them with a solution using a spray device, in which the sample (112) is sprayed several times in succession, a subsequent spray application only being carried out when the previously applied layer has dried .
Derartige Verfahren werden im Bereich der MALDI-Technik (Matrix Assisted Laser Desorption Ionisation) eingesetzt. Wird diese Technik auf Gewebeproben angewendet, wird sie als "MALDI Imaging" bezeichnet. Sie hat im Rahmen der bildgebenden Massenspektrometrie eine Vorreiterfunktion übernommen, die sich seit etwa 10 Jahren in der Krebsforschung, der pharmazeutischen Entwicklung und in der Proteinforschung etabliert hat. Der Vorteil der MALDI-Technik besteht darin, dass die Gewebeproben ohne großen Aufwand für die Untersuchung vorbereitet werden können, wobei nicht nur frisch gefrorene, sondern auch durch Formalin fixierte und in Silikon eingebettete und dadurch praktisch unbegrenzt haltbare Proben für diese neue und sehr empfindliche Untersuchungsmethode zugänglich sind, ebenso wie beispielsweise auch sehr altes Probenmaterial, das bis über 100 Jahre alt sein kann.Such methods are used in the MALDI technique (M atrix A ssisted L aser D esorption onisation I). When this technique is applied to tissue samples, it is called "MALDI Imaging". She has taken on a pioneering role in the field of imaging mass spectrometry, which has been established in cancer research, pharmaceutical development and protein research for around 10 years. The advantage of the MALDI technique is that the tissue samples can be prepared for the examination without great effort, whereby not only freshly frozen samples, but also samples fixed by formalin and embedded in silicone and thus practically indefinitely durable for this new and very sensitive examination method are accessible, as are, for example, very old sample material, which can be over 100 years old.
Die Grundlage der MALDI-Technik besteht darin, dass die Gewebeschnitte auf als Target bezeichnete Stahlplatten oder auf Objektträger aufgelegt werden, auf denen sie nach dem Trocknen dauerhaft haften und sich selbst fixieren. Anschließend werden die Schnitte mit einer als Matrix bezeichneten, meist polaren organischen Verbindung beschichtet, die die Eigenschaft hat, dass sie bei einer Bestrahlung mit Laserstrahlen im UV-Bereich das Licht absorbieren und dann durch den hohen Energieeintrag verdampfen. Dabei werden auch solche Stoffe der Gewebeprobe mit in die Dampfphase gerissen, die eigentlich gar nicht verdampfbar sind und bei normaler Erwärmung verbrennen würden. Durch die extrem rasche Verdampfung ist es aber möglich, derartige Proben zu verdampfen. In einem elektrischen Feld werden diese Moleküle ionisiert und von einem Massenspektrometer aufgenommen. In diesen Massenspektrometern werden Sie dann nach Ladung und Größe getrennt, wobei eine Software aus diesen zwei Parametern die Zusammensetzung entschlüsselt und identifiziert.The basis of the MALDI technique is that the tissue sections are placed on steel plates known as targets or on microscope slides, on which they adhere permanently and fix themselves after drying. Subsequently, the cuts are coated with a mostly polar organic compound called a matrix, which has the property that it changes when it is irradiated with laser beams absorb the light in the UV range and then evaporate due to the high energy input. In the process, substances in the tissue sample are also dragged into the vapor phase that are actually not vaporizable at all and would burn if heated normally. Due to the extremely rapid evaporation, however, it is possible to evaporate such samples. These molecules are ionized in an electric field and recorded by a mass spectrometer. In these mass spectrometers they are then separated according to charge and size, with software decoding and identifying the composition from these two parameters.
Es sind unterschiedliche Methoden bekannt, die Matrix auf die Gewebeproben aufzubringen, wobei sich die gewählte Methode nach den Analyten richten kann, die man untersuchen oder nachweisen möchte. Insbesondere ist zu erwähnen, dass Bio-Moleküle oder schwer verdampfbare Moleküle eine Extraktion und einen Einbau in die Kristallstruktur der Matrix benötigen, um in einer Weise verdampft werden zu können, dass sie anschließend mittels des Massenspektrometers ausgewertet werden können. Andere Verbindung wie Lipide brauchen als leicht verdampfbare Stoffe nur eine einfache Beschichtung ohne Extraktion. Für den letzteren Fall existiert mit der Sublimation mit einer Abscheidung der Matrix direkt aus der Gasphase bereits ein Auftragsverfahren mit einer sehr feinen Kristallstruktur, das sich aber nicht für die Extraktion eignet, da keine flüssige Phase vorhanden ist. Nachteilig ist auch, dass eine komplette Fläche auch neben der Probe und nicht nur die Probe selbst mit der Matrix überzogen wird, wodurch sich ein hoher Verbrauch an Matrixlösung einstellt, was insbesondere bei teuren Matrizes ins Gewicht fällt. Ohnehin eignen sich nur wenige Matrizes für diese Methode, während sie beispielsweise für Enzymlösungen nicht anwendbar ist.Different methods are known for applying the matrix to the tissue samples, and the method chosen can depend on the analytes that are to be examined or detected. In particular, it should be mentioned that bio-molecules or molecules that are difficult to vaporize require extraction and incorporation into the crystal structure of the matrix in order to be able to be vaporized in such a way that they can then be evaluated by means of the mass spectrometer. Other compounds such as lipids, being easily vaporized substances, only need a simple coating without extraction. In the latter case, sublimation with a deposition of the matrix directly from the gas phase already exists, an application process with a very fine crystal structure, which, however, is not suitable for extraction as there is no liquid phase. Another disadvantage is that a complete area is coated with the matrix next to the sample and not just the sample itself, which results in a high consumption of matrix solution, which is particularly important in the case of expensive matrices. In any case, only a few matrices are suitable for this method, while it cannot be used, for example, for enzyme solutions.
Um bei einer Beschichtung durch Sublimation dennoch eine Extraktion bewirken zu können, werden durch Sublimation beschichtete Objekte in einer wassergesättigten Umgebung 24 bis 72 Stunden aufbewahrt, wobei durch die Umgebungsfeuchtigkeit ein gewisses Maß an Extraktion stattfindet. Die extrem lange Probenvorbereitungszeit steht einer weiten Verbreitung dieser Methode entgegen.In order to still be able to effect an extraction with a coating by sublimation, objects coated by sublimation are stored in a water-saturated environment for 24 to 72 hours, with a certain amount of extraction taking place due to the ambient humidity. The extremely long sample preparation time prevents this method from being widely used.
Von den übrigen Sprühmethoden, beispielsweise der manuellen Sprühmethode, der Ultraschallversprühung oder der Elektrospray-Technik, ist die modifizierte Airbrush-Methode hervorzuheben, bei welcher ein optimierter Sprühkopf mit einer exakten Flüssigkeitsmenge pro Zeiteinheit beschickt wird. Ansonsten setzt ein derartiger Sprühkopf, wie der Name schon sagt, die Airbrush-Technik ein, bei welcher der Luftstrom nach dem Ejektor-Prinzip die Flüssigkeit mitreißt und fein verteilt. Eine derartige Methode eignet sich sehr gut für die Extraktion, wobei die erzielbaren Kristallgrößen nach dem Abtrocken der Matrix sich in einer Größenordnung von 30 bis 100 µm bewegen.Of the other spray methods, for example the manual spray method, ultrasonic spraying or electrospray technology, the modified airbrush method should be emphasized, in which an optimized spray head is charged with an exact amount of liquid per unit of time. Otherwise, such a spray head, as the name suggests, uses airbrush technology, in which the air flow according to the ejector principle entrains the liquid and distributes it finely. Such a method is very suitable for extraction, the crystal sizes that can be achieved after the matrix has dried off in the range of 30 to 100 μm.
Die Auflösung beim MALDI-Imaging wird durch die Kristallgröße der Matrix und den Laserdurchmesser bestimmt. Je kleiner die Auflösung dieser beiden Parameter, desto detailliertere Informationen erhält man über das Gewebe, wie z. B. Leber, Niere, Haut und Gehirn sowie allen anderen inneren Organen, bzw. ihrer Feinstruktur. Dies ermöglicht eine Fülle von neuen Informationen beispielsweise über die Entwicklung eines Krebses oder die neuen Verbindungen, die durch den Krebs neu entstehen, was wiederum eine frühe Erkennung der Krankheit ermöglicht.The resolution in MALDI imaging is determined by the crystal size of the matrix and the laser diameter. The smaller the resolution of these two parameters, the more detailed information can be obtained about the tissue, e.g. B. liver, kidney, skin and brain as well as all other internal organs or their fine structure. This enables a wealth of new information, for example about the development of a cancer or the new compounds that are created by the cancer, which in turn enables the disease to be detected early.
In der Pharmaforschung erhält man sehr detaillierte Informationen über die Verteilung eines Medikaments im lebenden Körper und deren Metabolite sowie die Ausscheidung aus dem Körper.Pharmaceutical research provides very detailed information about the distribution of a drug in the living body and its metabolites, as well as how it is excreted from the body.
Neuere Entwicklungen in der Lasertechnik haben für die Laserdurchmesser Dimensionen von ca. 1 µm geschaffen. Mit dieser Entwicklung können die bisherigen Kristallgrößen die durch die üblichen Sprühverfahren aufgetragen wird, nicht mithalten. Wie bereits erwähnt, liegt die Grenze in einer Größenordnung von 30 µm bis 100 µm als kleinste Kristallgröße, insbesondere dann, wenn eine Extraktion erforderlich ist, wenn größere Moleküle wie Peptide oder Proteine in einem Molekulargewichtsbereich von >100 bis über 100 000 Dalton erfasst werden sollen. Wenn jedoch die Kristallgröße in einer Größenordnung von 30 µm aufwärts liegt, hat dies zur Folge, dass bei einem Auftreffen des Laserstrahles selbst dann, wenn dieser einen Durchmesser von ca. 1 µm hat, der komplette Kristall verdampft, so dass die Auflösung in diesem Fall durch die Matrixkristallgröße und nicht durch den Laserdurchmesser bestimmt wird.More recent developments in laser technology have created dimensions of approx. 1 µm for the laser diameter. The previous crystal sizes that are applied using the usual spraying methods cannot keep up with this development. As already mentioned, the limit is on the order of 30 µm to 100 µm as the smallest crystal size, especially when extraction is required when larger molecules such as peptides or proteins in a molecular weight range of> 100 to over 100,000 Daltons are to be detected . If, however, the crystal size is in the order of 30 µm upwards, this has the consequence that when the laser beam hits, even if it has a diameter of approx. 1 µm, the complete crystal evaporates, so that the resolution in this case is determined by the matrix crystal size and not by the laser diameter.
Beispiele für derartige Verfahren sind in der
Die Aufgabe der vorliegenden Erfindung besteht darin, ein Verfahren der eingangs genannten Art dahingehend zu verbessern, dass eine Bildung kleinerer Kristalle der Maxtrix in der Beschichtung nach dem Abtrocknen ermöglicht ist. Die Beschichtung soll auch möglichst homogen ausgebildet sein, um die Erfassung der Moleküle nicht nur qualitativ sondern auch semi-quantitativ oder quantitativ zu ermöglichen.The object of the present invention is to improve a method of the type mentioned at the outset such that smaller Maxtrix crystals can be formed in the coating after it has dried. The coating should also be designed as homogeneously as possible in order to to enable the detection of the molecules not only qualitatively but also semi-quantitative or quantitative.
Gelöst wird die Aufgabe durch ein Verfahren der eingangs genannten Art, bei welchem die erste aufgetragene Schicht mit einer kleineren Menge an Lösung pro Fläche besprüht wird, die Menge bei den nachfolgenden Schichten erhöht werden, bis eine bestimmte Menge an Lösung pro Fläche erreicht wird, die für die letzte Schicht oder für mehrere Schichten mehrfach wiederholt aufgetragen wird.The object is achieved by a method of the type mentioned at the outset, in which the first applied layer is sprayed with a smaller amount of solution per area, the amount in the subsequent layers is increased until a certain amount of solution per area is reached is applied repeatedly for the last layer or for several layers.
Es hat sich gezeigt, dass sich durch das mehrfache Auftragen der Sprühlösung unter zwischenzeitlichem Abtrocknen unerwünschte Effekte vermeiden lassen, die bisher bei zu großer Flüssigkeitsmenge auftraten, nämlich dass kleinere Moleküle ihre ursprünglichen Lage verlassen und anfangen in verschiedene Richtungen zu diffundieren, so dass am Ende die Exaktheit der Lageinformationen bei der Massenspektroskopie verloren geht. Durch das schichtweise Auftragen kann eine 1.-3., recht dünne Schicht in der Art einer Fixierschicht eingesetzt werden, die die Lageinformationen sicherstellt und zum anderen eine Art saugfähige Unterlage für weitere Schichten bildet, die dann bei geeigneter Menge an Matrixlösung auch dazu geeignet sind, die gewünschte Extraktion größerer Moleküle in der gewünschten Art und Weise in die Matrixstruktur zu erreichen. Entsprechend sieht eine bevorzugte Ausführungsform des Verfahrens vor, dass die erste aufgetragene Schicht mit einer kleineren Menge an Lösung pro Fläche besprüht wird, die Menge bei den nachfolgenden Schichten erhöht wird, bis eine bestimmte Menge an Matrix pro Fläche erreicht wird, und diese Menge an Lösung pro Fläche für die letzten Schichten mehrfach wiederholt aufgetragen wird. Eine typische Anzahl von Schichten liegt im Bereich zwischen 5 bis 10, beispielsweise 8 Schichten, so dass sich insgesamt eine zweckmäßige Dicke der Matrix ergibt, die bei Bestrahlung mit dem Laser dann lokal auf einer auf die erreichte Kristallgröße reduzierten Fläche verdampft. Die ersten 1-3 Schichten, die mit der kleineren Menge an Lösung pro Fläche besprüht werden, trocknen dabei sehr schnell ab, so dass keine Lageveränderungen kleiner Moleküle zu befürchten sind.It has been shown that by repeatedly applying the spray solution while drying it off in the meantime, undesired effects can be avoided that previously occurred when the amount of liquid was too large, namely that smaller molecules leave their original position and start to diffuse in different directions, so that in the end the The accuracy of the positional information is lost in mass spectroscopy. Due to the layer-by-layer application, a 1st-3rd, very thin layer in the form of a fixing layer can be used, which ensures the position information and on the other hand forms a kind of absorbent underlay for further layers, which are then also suitable for this with a suitable amount of matrix solution to achieve the desired extraction of larger molecules in the desired manner into the matrix structure. Accordingly, a preferred embodiment of the method provides that the first applied layer is sprayed with a smaller amount of solution per area, the amount is increased in the subsequent layers until a certain amount of matrix per area is reached, and this amount of solution is applied several times per area for the last layers. A typical number of layers is in the range between 5 to 10, for example 8 Layers, so that overall an appropriate thickness of the matrix results, which when irradiated with the laser then evaporates locally on an area reduced to the crystal size achieved. The first 1-3 layers, which are sprayed with the smaller amount of solution per area, dry very quickly, so that no changes in the position of small molecules are to be feared.
Beispielsweise kann von einer ersten Menge an Lösung pro Fläche für die erste Schicht ausgehend die Menge beim Auftragen der zweiten Schicht verdoppelt und beim Auftragen der dritten Schicht verdreifacht werden, bis bei der dritten bis sechsten Schicht ein Maximum erreicht wird, das für alle nachfolgenden Schichten zur Anwendung kommt.For example, starting from a first amount of solution per area for the first layer, the amount can be doubled when applying the second layer and tripled when applying the third layer until a maximum is reached in the third to sixth layers that is applicable to all subsequent layers Application comes.
Da in der Regel der mittels einer in üblicher Art und Weise mit einer zweckmäßigen Geometrie des Sprühkopfes ausgebildeten Sprühvorrichtung eine Fläche mit einem Durchmesser von z.B. 2 mm besprüht werden kann, ist es bei entsprechend größeren Proben zweckmäßig, dass diese unter Verwendung einer Vorrichtung mit einer Verstellbarkeit in der X- und Y-Richtung zum Aufbringen einer Schicht zeilenweise besprüht wird. Nach dem zeilenweisen Besprühen der Probe und entsprechen vollständigem Aufbringen einer Schicht, fährt der Sprühkopf dann in die Ausgangslage zurück und wiederholt den Auftrag zeilenweise für die nächste Schicht.Since, as a rule, the spray device formed in the usual way with an appropriate geometry of the spray head has an area with a diameter of e.g. 2 mm can be sprayed, it is expedient for correspondingly larger samples that this is sprayed line by line using a device with adjustability in the X and Y directions for applying a layer. After the sample has been sprayed line by line and a layer has been completely applied, the spray head then moves back to the starting position and repeats the application line by line for the next layer.
Eine bevorzugte Weiterbildung der Erfindung sieht die Verwendung einer Sprühvorrichtung vor, bei welcher der Flüssigkeitsauslass am Ende einer Kapillarleitung vorgesehen ist, die beispielsweise mittig über den Gasauslass hinaus hervorsteht.A preferred further development of the invention provides for the use of a spray device in which the liquid outlet is provided at the end of a capillary line which, for example, protrudes centrally beyond the gas outlet.
Es hat sich gezeigt, dass mit einer solchen Sprühvorrichtung bedingt durch den über den Gasauslass überstehenden Flüssigkeitsauslass eine besonders feine Tröpfchengröße erreichbar ist, die letzten Endes zu einer Matrixschicht auf der Probe führt, die nach dem Trocknen eine kleinere Kristallgröße bei homogener Schichtbildung aufweist. Überraschenderweise ergibt sich der Effekt gerade dadurch, dass der Gasauslass im Verhältnis zum Flüssigkeitsauslass zurückgesetzt vorgesehen ist, so dass das Gas bis zum Erreichen der Flüssigkeit verwirbelt wird und dieses nicht mehr, wie bisher, nach dem Ejektorprinzip mitreißt, sondern dass die feine Tröpfchenbildung in dem verwirbelten Gas ausgebildet wird.It has been shown that with such a spray device, due to the liquid outlet protruding above the gas outlet, a particularly fine droplet size can be achieved, which ultimately leads to a matrix layer on the sample which, after drying, has a smaller crystal size with homogeneous layer formation. Surprisingly, the effect arises precisely from the fact that the gas outlet is set back in relation to the liquid outlet, so that the gas is swirled until it reaches the liquid and no longer carries it along according to the ejector principle, as before, but rather that the fine droplet formation in the swirled gas is formed.
Als Gase eignen sich im übrigen Luft oder auch Stickstoff, je nach Art der zu versprühenden Matrixlösung und zu untersuchenden Probe.In addition, air or nitrogen are suitable as gases, depending on the type of matrix solution to be sprayed and the sample to be examined.
In einer besonders bevorzugten Ausführungsform der Erfindung ist vorgesehen, dass das Ende der Kapillarleitung an seinem Außenumfang verjüngt ist, vorzugsweise konisch verjüngt ist. Es hat sich gezeigt, dass durch diese Maßnahme eine besonders feine Tröpfchenbildung ermöglicht ist, weil durch die Verjüngung, vorzugsweise in konischer Form, der verwirbelte Gasstrom besonders effektiv in Richtung des Flüssigkeitsauslasses umgelenkt wird.In a particularly preferred embodiment of the invention it is provided that the end of the capillary line is tapered on its outer circumference, preferably is tapered conically. It has been shown that this measure enables particularly fine droplet formation because the tapering, preferably in a conical shape, deflects the swirled gas flow particularly effectively in the direction of the liquid outlet.
In jedem Fall erfolgt unabhängig von der Ausbildung des Endes der Kapillarleitung eine aktive Zufuhr der Matrixlösung z. B. mittels einer geeigneten Pumpe, die in der Lage ist, die sehr kleinen Flüssigkeitsmengen zu fördern, die beim Auftrag der Schichten benötigt werden. Auf typische Flussmengen wird später noch im Rahmen der Ausführungsbeispiele eingegangen.In any case, regardless of the design of the end of the capillary line, there is an active supply of the matrix solution z. B. by means of a suitable pump that is able to deliver the very small amounts of liquid that are required when applying the layers. Typical flow rates will be discussed later in the context of the exemplary embodiments.
In einer bevorzugten Ausführungsform ist der Gasauslass durch einen Ringspalt zwischen der Außenwandung der Kapillarleitung und einem Führungsschlauch oder -rohr ausgebildet. Der Führungsschlauch oder das Führungsrohr dient dabei gleichzeitig zur Stabilisierung der empfindlichen Kapillarleitung, die in der Regel als Quarzkapillare ausgebildet ist.In a preferred embodiment, the gas outlet is formed by an annular gap between the outer wall of the capillary line and a guide hose or tube. The guide tube or the guide tube also serves to stabilize the sensitive capillary line, which is usually designed as a quartz capillary.
Der Führungsschlauch oder das Führungsrohr, das beispielsweise aus PEEK (Polyethereetherketon) bestehen kann, kann wiederum von einem größeren Durchmesser stabilisiert sein, wobei letzten Endes das Führungsrohr und/oder die Kapillarleitung vorzugsweise in einem Gehäuse gasdicht gehalten sind. Das Gehäuse hat zum Einen die Aufgabe, die Position des Düsenkopfes exakt zu definieren, zum anderen besitzt das Gehäuse vorzugsweise einen Gasanschluss für die Gaszufuhr, wobei in dem Gehäuse ein Gasdurchlass in einem Ringspalt zwischen dem Führungsrohr oder dem Führungsschlauch und der Kapillarleitung vorgesehen ist. Auf diese Weise kommt dem Gehäuse die Funktion zu, den Gasstrom von der Gaszufuhr in den Ringspalt zwischen den Führungsschlauch oder dem Führungsrohr und der Kapillarleitung zu leiten.The guide tube or the guide tube, which can for example consist of PEEK (polyether ether ketone), can in turn be stabilized by a larger diameter, with the guide tube and / or the capillary line preferably being held gas-tight in a housing. On the one hand, the housing has the task of precisely defining the position of the nozzle head and, on the other hand, the housing preferably has a gas connection for the gas supply, with a gas passage in the housing in an annular gap between the guide tube or the guide tube and the capillary line. In this way, the function of the housing is to guide the gas flow from the gas supply into the annular gap between the guide tube or the guide tube and the capillary line.
In der Regel durchragt die Kapillarleitung des Gehäuse und ist an ihrem anderen Ende mit einer bereits erwähnten Förderpumpe für die Flüssigkeitszufuhr verbunden, wobei anstelle einer Pumpe auch ein Dispenser mit einer geeigneten hohen Auflösung, beispielsweise 24.000 Schritten pro Spritzenfüllung eingesetzt werden kann. Um den rückwärtigen Austritt der Kapillarleitung aus dem Gehäuse gasdicht zu verschließen, ist vorzugsweise auf der dem Sprühkopf abgewandten Seite des Gehäuses ein Stopfen vorgesehen, der das Gehäuse gegen die Außenwandung der Kapillarleitung gasdicht verschließt.As a rule, the capillary line protrudes through the housing and is connected at its other end to an already mentioned feed pump for the supply of liquid, whereby a dispenser with a suitable high resolution, for example 24,000 steps per syringe filling, can be used instead of a pump. In order to seal the rear exit of the capillary line from the housing in a gastight manner, a plug is preferably provided on the side of the housing facing away from the spray head, which plug closes the housing in a gastight manner against the outer wall of the capillary line.
Um die bereits angesprochene Positionierung des Düsenkopfes besonders exakt vornehmen zu können, verfügt das Gehäuse vorzugsweise über einen Absatz, der in einem definierten Abstand zum Flüssigkeitsauslass angeordnet ist. Der Flüssigkeitsauslass definiert mit seiner exakten Lage die Position des Düsenkopfes.In order to be able to carry out the already mentioned positioning of the nozzle head particularly precisely, the housing preferably has a shoulder which is arranged at a defined distance from the liquid outlet. The exact position of the liquid outlet defines the position of the nozzle head.
Vorzugsweise ist der Düsenkopf an einer Verfahrvorrichtung gehalten, die ein Verfahren der Position des Düsenkopfes in der X-, Y- und/oder Z-Richtung ermöglicht. Grundsätzlich ist die Verfahrmöglichkeit in X- und Y-Richtung wichtiger, da ggf. auch mit einer festen Position, d. h. einem festen Abstand des Flüssigkeitsauslasses zur Probenoberfläche gearbeitet werden kann. Der Düsenkopf wird beispielsweise mit Hilfe des zuvor beschriebenen Absatzes an dem Gehäuse in eine definierte Position bezüglich der Gewebeoberfläche gebracht.The nozzle head is preferably held on a displacement device which enables the position of the nozzle head to be moved in the X, Y and / or Z directions. Basically, the ability to move in the X and Y directions is more important, since it may also have a fixed position, i.e. H. a fixed distance between the liquid outlet and the sample surface can be used. The nozzle head is brought into a defined position with respect to the tissue surface, for example with the aid of the paragraph described above on the housing.
Nachfolgend wird anhand der beigefügten Zeichnungen näher auf ein Ausführungsbeispiel der Erfindung eingegangen. Es zeigen:
- Fig. 1
- einen Schnitt einer Gesamtansicht eines Sprühkopfes einer Sprühvorrichtung;
- Fig. 2
- eine Detailansicht der Austrittsbereiche des Sprühkopfes;
- Fig. 3
- eine schematische Ansicht einer Sprühvorrichtung mit verstellbarem Sprühkopf.
- Fig. 1
- a section of an overall view of a spray head of a spray device;
- Fig. 2
- a detailed view of the exit areas of the spray head;
- Fig. 3
- a schematic view of a spray device with an adjustable spray head.
In
Die Quarzkapillarleitung 14 ist durch ein Gehäuse 18 hindurch geführt, das an seinem rückwärtigen Ende mit Hilfe einer Überwurfmutter 20 verschlossen ist, wobei die Überwurfmutter 20 in einem Gewinde 22 in das Gehäuse 18 eingreift und das Gehäuse 18 gegen den Außenumfang der Quarzkapillare 14 abdichtet. Das Gehäuse selbst ist gestuft mit einem Absatz 24 ausgebildet, auf dessen Funktion später noch näher eingegangen wird.The
Das Gehäuse ist innenseitig mit einer mittigen Bohrung 26 versehen, die mit einem radialen Gasanschluss 28 in Verbindung steht, der mit einer Gaszufuhr (nicht gezeigt) zu verbinden ist. Die Gaszufuhr sorgt durch geeignete Pumpmittel für die Zufuhr von Luft oder einem anderen geeigneten Gas wie z. B. Stickstoff, unter einem Druck von üblicherweise 2 bis 3 Bar, der im Betrieb konstant gehalten wird, wobei auch andere Druckwerte realisierbar sind.The housing is provided on the inside with a
An dem der Überwurfmutter 20 gegenüberliegenden Ende des Gehäuses 18 sitzt in einem erweiterten Bohrungsabschnitt 30 ein erster Führungsschlauch 32, der auch als Führungsrohr ausgebildet sein kann, innerhalb welchem ein weiterer Führungsschlauch 34 druckdicht eingelassen ist. Der zweite Führungsschlauch 34 umschließt die Quarzkapillare 14 an ihrem Außenumfang mit einem Ringspalt, d. h. zwischen dem zweiten Führungsschlauch 34 und der Quarzkapillare besteht ein Gasdurchlass zwischen der Bohrung 26 des Gehäuses 10 und einem Gasauslass 36 (siehe
Wie aus
Der in
Normalerweise erfolgt das Besprühen des Gewebes 112 in der Weise, dass der Düsenkopf 10 die zu besprühende Fläche zeilenweise abfährt, da in der Regel der durch den Sprühstrahl erfasste Bereich kleiner ist als die Fläche des zu besprühenden Gewebes. Typischerweise erfasst die versprühte Matrixlösung bei einem zeilenweisen Verfahren eine streifenförmige Fläche von einer Breite von ungefähr 2 mm.The
Nachfolgend wird ein Ausführungsbeispiel im Detail beschreiben, bei dem einige Parameter explizit genannt werden, auch wenn sie sich im Rahmen der Erfindung in einem weiten Bereich bewegen können.In the following, an exemplary embodiment is described in detail in which some parameters are named explicitly, even if they can vary within a wide range within the scope of the invention.
Es wurde eine Matrixlösung versprüht, die im Bereich des Düsenkopfes 10 mit einem unter einem konstanten Druck von 2,5 bar zugeführten Luftstrom vermischt worden ist. Der Außendurchmesser der gewählten Quarzkapillare betrug 280 µm, wobei zwischen dem zweiten Führungsschlauch 34 und dem Außendurchmesser der Quarzkapillare 14 ein Ringspalt mit einer Höhe von 60 µm vorgesehen war, d. h. der Innendurchmesser des aus PEEK gefertigten zweiten Führungsschlauches betrug 400 µm. Die Quarzkapillare 14 besaß einen inneren Kanal 42 mit einem Durchmesser von 75 µm.A matrix solution was sprayed which was mixed in the area of the
Unter einem fest eingestellten Abstand von 40 mm zwischen dem Flüssigkeitsaustritt 16 und der Oberfläche des Targets 110, wurde letzteres zeilenweise abgefahren, bis eine gewünschte Fläche vollständig mit der Matrixlösung besprüht worden war. Dabei wird bei der gewählten Düsengeometrie und dem gewählten Gasdruck ein Bereich von ca. 2 mm direkt besprüht, wobei die Zeilenabstände so gewählt sind, dass eine Überdeckung benachbarter besprühter Zeilen nur minimal vorhanden ist, um abweichende Schichtdicken im Überlappungsbereich zu vermeiden.With a fixed distance of 40 mm between the
Beim Aufbringen einer ersten Schicht, ist eine Flüssigkeitszufuhr von 10 µl pro Minute gewählt worden, wobei die Verfahrgeschwindigkeit 200 cm pro Minute betrug.When applying a first layer, a liquid supply of 10 µl per minute was selected, the travel speed being 200 cm per minute.
Nach dem Aufbringen der ersten Schicht, wurde eine zweite Schicht durch Versprühen aufgebracht, die bei ansonsten gleichen Verfahrensparametern unter ein Verdopplung der Flüssigkeitszufuhr auf 20 µl pro Minute durchgeführt worden ist. Eine dritte Schicht wurde mit 30 µl pro Minute, eine vierte mit 40 µl pro Minute aufgebracht, wobei insgesamt 8 Schichten aufgebracht wurden, die ab der vierten Schicht alle unter einer Flüssigkeitszufuhr von 40 µl pro Minute aufgebracht worden sind.After the first layer had been applied, a second layer was applied by spraying, which was carried out with otherwise the same process parameters while doubling the liquid supply to 20 μl per minute. A third layer was applied at 30 µl per minute, a fourth at 40 µl per minute, a total of 8 layers being applied, all of which were applied from the fourth layer onwards with a liquid supply of 40 µl per minute.
Eine elektronenmikroskopische Untersuchung der Schicht ergab, dass sich Matrixkristalle einer sehr konstanten Kristallgröße im Bereich zwischen 130 bis 140 nm ausgebildet hatten. Mit derartigen Kristallgrößen eignete sich die Probe hervorragend für die weitere Untersuchung im Rahmen der MALDI-Technik der neusten Generation mit einem Laser, dessen Lichtstrahl auf 1 µm Größe fokussiert war. Durch die kleinen Kristalle ist die Auflösung der massenspektrometrischen Untersuchung durch den extrem kleinen Querschnitt des Laserstrahls von 1 µm definiert, was unterhalb der Größe einer menschlichen Zelle von bis zum 10 µm liegt, so dass im Rahmen dieser Untersuchung das Gewebe nach einzelnen Zellen analysiert werden konnte.An electron microscopic examination of the layer showed that matrix crystals with a very constant crystal size in the range between 130 and 140 nm had formed. With such crystal sizes, the sample was ideally suited for further investigation in the context of the latest generation of MALDI technology with a laser whose light beam was focused to a size of 1 µm. The mass spectrometric investigation is resolved by the small crystals defines the extremely small cross-section of the laser beam of 1 µm, which is below the size of a human cell of up to 10 µm, so that the tissue could be analyzed for individual cells within the scope of this investigation.
Das beschriebene Ausführungsbeispiel ist mit seinen Eckdaten nicht zwingend. Insbesondere können die Dimensionen der Quarzkapillare und des Führungsschlauches deutlich abweichen, aber auch die Flüssigkeitsmenge kann je nach Art zu versprühenden Matrixlösung deutlich von den genannten Werten abweichen. Auch bei der Gaszufuhr sind Abweichungen von dem gewählten Druck möglich, um je nach gewünschter Untersuchung entsprechend unterschiedliche Schichten aufbringen zu können.The exemplary embodiment described, with its key data, is not mandatory. In particular, the dimensions of the quartz capillary and the guide tube can differ significantly, but the amount of liquid can also differ significantly from the stated values depending on the type of matrix solution to be sprayed. Deviations from the selected pressure are also possible with the gas supply in order to be able to apply different layers depending on the desired examination.
Claims (11)
- Method for coating samples (112) by spraying with a solution using a spraying device, in which the sample (112) is sprayed several times in succession, a subsequent spray application being carried out only once the previously applied coat has dried, characterised in that the first applied coat is sprayed with a smaller quantity of solution per surface area, the quantity is increased in the subsequent coats, until a specified quantity of solution per surface area is reached, which is applied for the last coat or repeatedly applied for several coats.
- Method according to claim 1, characterised in that a first quantity of solution per surface area for the first coat is doubled in the application of the second coat, trebled in the application of the third coat and, where appropriate, further increased in a further coat up to a maximum, which is used for application of all subsequent coats.
- Method according to claim 1 or 2, characterised in that for the application of each coat the sample is sprayed in lines.
- Method according to any one of claims 1 to 3, characterised by the use of a spraying device for spraying samples (112) with a solution, wherein a liquid feed for the dosed feeding of the solution and a gas feed are provided, which are connected by supply lines to a nozzle head (10), which has a gas outlet (36) and a liquid outlet (16), for spraying of the solution, and in that the liquid outlet (16) is provided at the end of a capillary line (14), which projects beyond the gas outlet (36).
- Method according to claim 4, characterised by the use of a spraying device in which the end of the capillary line (14) is tapered at its outer circumference.
- Method according to claim 4 or 5, characterised by the use of a spraying device in which the gas outlet (36) is formed by an annular gap between an outer circumference of the capillary line (14) and a guide hose (34) or guide tube.
- Method according to any one of claims 4 to 6, characterised by the use of a spraying device in which the guide hose (34) and/or the capillary line (14) are held in a housing (18) in a gas-tight manner.
- Method according to claim 7, characterised by the use of a spraying device, the housing (18) of which has a gas connector (28) for the gas feed and in the housing a gas outlet is provided in an annular gap between the guide hose (34) or tube and the capillary line (14).
- Method according to claim 7 or 8, characterised by the use of a spraying device in which the housing (10), on its side that faces away from the liquid outlet (16), is provided with a threaded stopper (20) which closes the housing (10) against the outer wall of the capillary line (14) in a gas-tight manner.
- Method according to any one of claims 7 to 9, characterised by the use of a spraying device in which the housing has a shoulder (24), which is arranged at a defined distance from the liquid outlet (16).
- Method according to any one of claims 4 to 9, characterised by the use of a spraying device in which the nozzle head (10) is held on a displacing device (100), which enables displacement of the position of the nozzle head (10) in the X-, Y- and/or Z-direction.
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US5772964A (en) * | 1996-02-08 | 1998-06-30 | Lab Connections, Inc. | Nozzle arrangement for collecting components from a fluid for analysis |
DE20210784U1 (en) * | 2001-11-14 | 2003-11-27 | CARBOTEC Gesellschaft für instrumentelle Analytik mbH | Electrostatic spraying unit used in mass spectrometry directs gas flow to avoid direct influence on tip or capillary |
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2015
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DE102015113170A1 (en) | 2017-02-16 |
WO2017025205A1 (en) | 2017-02-16 |
US20180236470A1 (en) | 2018-08-23 |
EP3334537A1 (en) | 2018-06-20 |
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